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Microbial Genomics

Microbial Genomics. Topics Describe the new area of genomics Outline the rapid progress in genomic sequencing Describe the analysis of sequences - bioinformatics Show the use of genomics in the study of microbes

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Microbial Genomics

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  1. Microbial Genomics Topics • Describe the new area of genomics • Outline the rapid progress in genomic sequencing • Describe the analysis of sequences - bioinformatics • Show the use of genomics in the study of microbes • Use the sequence of a human pathogenEscherichia coli O157:H7 to illustrate the above points Ref: Perna et al. (2001) Nature 409:529 (USA) • Relevant to next lectures. Dr M. D-S, 2007

  2. Microbial genome sequences Genbank (NCBI), Bethesda, Maryland, USA 2007: 481 - completed microbial genomes 2006: 3192003: 112 Sizes range from 0.58 - over 9 Mb Genbank - main genomic database There is some duplication... Dr M. D-S, 2007

  3. Genomics - the study of entire genomes of organisms • assumes the entire sequence of at least one representative example has been determined • includes study of all the genes and gene products and non-coding regions • includes study of genome organisation and evolution Dr M. D-S, 2007

  4. The explosion of ‘-ome’ and ‘-omics’ words • Functional genomics • Proteome • Transcriptome • Metabolome, Glycome, Lipidome e.g. a recent journal article with the title: “Functional genomics by integrated analysis of metabolome and transcriptome of Arabidopsis” Dr M. D-S, 2007

  5. Genomics What can microbial genomics tell us ? • Full gene complement of the cell • Complete description of cell metabolism • How genomes are structured • Virulence genes • Potential drug targets • Gene flow between cells (evolution) Dr M. D-S, 2007

  6. Genome Sequencing:Twomethods • 1. Sanger di-deoxy sequencing (using fluorescently labelled ddNTPs) on cloned DNA templates. • 2. Pyro-sequencing method on 454 machine using uncloned DNA templates Dr M. D-S, 2007

  7. Genome Sequencing:Twomethods 1. Sanger di-deoxy sequencing (using fluorescently labelled ddNTPs) on cloned DNA templates. ‘Shotgun’ strategy. • Dye-terminator chemistry, ABI sequencing apparatus, commercial software for handling seq. data Dr M. D-S, 2007

  8. Genomic sequencing methods Shear DNA & isolate fragments about 2kb chDNA Clone thousands of fragments into plasmid vector (library). Prepare DNA for sequencing Dr M. D-S, 2007

  9. Dideoxy chain termination http://www.plattsburgh.edu/acadvp/artsci/biology/bio401/DNASeq.html Dr M. D-S, 2007

  10. Sequence: methods section - Applied Biosystems Inc (ABI) latest sequencing machine, PE 3700 Capillary electrophoresis 96 capillaries at a time Robotically loaded and run (24hr) How many bp can it do in a day?? - each run is 2hr, get 600-1000 nt per capillary, 96 capillaries/run + Dr M. D-S, 2007

  11. Sequence: methods section Applied Biosystems Inc (ABI) latest sequencing machine, PE 3700 How many bp can it do in a day?? - each run is 2hr, about 800bp each lane, 96 lanes = 24/2  800  96 = 921,000 Or about 1 Mb /machine/day Dr M. D-S, 2007

  12. Top of capillary tubes Sequence data - Laser scanning of the 96 capillary tubes identifies the colour and positions of the closely spaced bands of ssDNA. + TAATCATGGTC.... Dr M. D-S, 2007

  13. Shotgun sequencing: how much do you need to do? ~ 1 Mb /machine/day Want both strands, good sequence for both, random coverage means you will need 6-8x genome size in sequence data Speed makes it efficient? Counter argument is the difficulty in linking up reads, particularly when genomes have long repeat sequences. Dr M. D-S, 2007

  14. Genome Sequencing:Twomethods In the E.coliO157:H7genome sequence paper by Perna et al., there were 2 gaps remaining in the genome sequence! They couldn’t complete it. “Extended exact matches pose a significant assembly problem.” ?? Dr M. D-S, 2007

  15. Repeat sequences, eg. Prophage genomes Nearly identical prophage sequences at 3 locations on genome, all > 2000 nt What sequences do you observe when inside a prophage genome? Dr M. D-S, 2007

  16. Repeat sequences, eg. Prophage genomes Nearly identical prophage sequences at 2 locations on genome What sequences do you see going across the borders of prophages? Dr M. D-S, 2007

  17. Repeat sequences, eg. Prophage genomes Nearly identical prophage sequences at 2 locations on genome What information do you need to place the repeats properly? Dr M. D-S, 2007

  18. Genome Sequencing:Twomethods • 1. Sanger di-deoxy sequencing (using fluorescently labelled ddNTPs) on cloned DNA templates. • 2. Pyro-sequencing method on 454 machine using uncloned DNA templates Dr M. D-S, 2007

  19. The 454 machines: the next revolution www.454.com

  20. The 454 machines: the next revolution 40 million bases/5.5 hr www.454.com

  21. The 454 machines: the next revolution 40 million bases/5.5 hr DNA immobilised on micro-beads Positioned in wells of special tray (44um diameter, 1.2 million per chip) Sequencing enzymes on smaller beads. Only one DNA-bead can fit in each well Each bead has only one DNA fragment attached, so will give unique sequence. www.454.com

  22. The 454 machines: the next revolution When a base is incorporated (by DNA polymerase), light is emitted, and the light detected under each well. www.454.com

  23. The 454 machines: the next revolution 40 million bases/5.5 hr When a base is incorporated (by DNA polymerase), light is emitted, and the light detected under each well. If there are multiple bases, the light is proportional to the number. Chain lengths of 200 nt are possible. With 200,000 wells, and 200nt/well, then 40 million bases can be sequenced. www.454.com

  24. Genomics • Papers filled with JARGON. Mainly genetic terms. Some terms are relatively new (eg. replichore) • Use the E.coli paper example, stopping to investigate each new term or concept • Emphasise the uses of this data, and the future of genomic research. Dr M. D-S, 2007

  25. What do you know about microbial genomes ? Exercise: Think of a typical bacterial genome, like that of E.coli and - • Sketch the genome and the most significant features you know about it (as a whole genome, not individual genes) • Jot down what you think the main selective pressures are on it Dr M. D-S, 2007

  26. Escherichia coli genome • Circular, ~ 4.6 Mb • Ori and Ter, bidirectional replication • Replichores about equal oriC ter Dr M. D-S, 2007

  27. Replichore ‘balance’ ? • If you move oriC relative to Ter, the growth rate of E. coli K-12 is reduced. • Chromosomal inversions around the origin or termination of replication are usually symmetrical, conserving the replichore balance. Hill, C. W., and J. A. Gray. 1988. Effects of chromosomal inversion on cell fitness in Escherichia coli K-12. Genetics 119:771–778. Eisen, J. A., J. F. Heidelberg, O. White, and S. L. Salszberg. 2000. Evidence for symmetric chromosomal inversions around the replication origin in bacteria. Genome Biol. 1:0011.1–0011.9 Dr M. D-S, 2007

  28. E.coli genome - global features • Gene dosage • Gene direction relative to ori • Recombination/inversion rates vary around chromosome Dr M. D-S, 2007

  29. Gene Dosage • Genes near the origin of replication will almost always be in multiple copy compared to genes near the terminus • So the position of a gene relative to the origin will affect its expression, and the regulatory systems would have evolved to accommodate for the gene dosage effect. • So what would happenif you moved genes ? oriC ter Dr M. D-S, 2007

  30. Gene Direction • What happens when a DNA pol meets an RNA pol going in the opposite direction? RNAPolymerase DNAPolymerase Dr M. D-S, 2007

  31. Gene Direction • What happens when a DNA pol meets an RNA pol going in the opposite direction? RNAPolymerase DNAPolymerase This is better…. Dr M. D-S, 2007

  32. Gene Direction ori A preference for genes to be on ONE strand of the replichore, so that the direction of transcription and replication are the same. This bias may have other implications. Dr M. D-S, 2007

  33. Recombination/inversions • Genomes often have large repeated sequences, eg. ribosomal RNA gene clusters (16S-23S-5S), or phage genomes. • Such repeats allow large inversions of DNA segments or recombination between chromosomes Dr M. D-S, 2007

  34. Inversion via repeated sequences Homologous recombination between rRNA genes Dr M. D-S, 2007

  35. origin GC-skew Chi sequences terminus Dr M. D-S, 2007

  36. Genomics: What is GC-skew ? Systematic bias in base composition of one strand as you go around the genome origin [G-C] [G+C] GC skew ter ter genome Dr M. D-S, 2007

  37. GC-skew of genomes Dr M. D-S, 2007

  38. Compositional bias: Leading strand enriched in G/T (keto) Lagging strand enriched in C/A (amino) WHY? Perhaps due to deamination of exposed C’s in the leading strand, producing C>T mutations. Theory only. GC-skew of genomic DNA Dr M. D-S, 2007

  39. origin GC-skew Chi sequences terminus Dr M. D-S, 2007

  40. E.coliO157:H7-K12genome comparison: Chi sequences GCTGGTGG • Sequence recognised (and cut) by the RecBC enzyme • Promotes homologous recombination (by RecA) Dr M. D-S, 2007

  41. Lateral Gene Transfer (LGT) • Literally, the natural transfer of genetic material between different organisms (species, genera, etc) • Doesn’t say how the DNA was transferred or integrated, or where it came from. • Does imply that the DNA can be identified as ‘foreign’ • Since DNA doesn’t have a ‘made in X’ sticker, how can the ‘foreignness’ be identified? …. Ideas?…. Dr M. D-S, 2007

  42. Lateral Gene Transfer (LGT) Known mechanisms of DNA transfer between bacteria:- • Transduction • transducing bacteriophages introduce host DNA, and this recombines with the genome • Transformation • DNA uptake from the surroundings, and recombination. • Conjugation • natural transfer method, sex pilus, one-way transfer, recombination. + - Dr M. D-S, 2007

  43. Prophage Bacteriophages that are temperate (as compared to lytic) can exist inside host cells in a stable and relatively inactive state as prophages. • The host cell, with a prophage, is called a lysogen. • Some prophages express virulence determinants, such as toxins ( = lysogenic conversion). eg. Shiga toxin • Some prophages exist as plasmids, but most integrate into the genome. • If the prophage becomes damaged…. ? Dr M. D-S, 2007

  44. E.coli genome sequences STRAIN SIZE DATE E.coli K12 4639221 bp, Oct 13 1998 E.coli O157:H7 (USA) 5528970 bp, Jan 25, 2001 E.coli O157:H7 (Japanese) 5498450 bp, Mar 7, 2001 *about 4.1Mb in common Data from NCBI: http://www.ncbi.nlm.nih.gov:80/PMGifs/Genomes/eub.html Dr M. D-S, 2007

  45. A B X C D A B C D A B C D A B C D E.coli O157:H7 - K12 genome comparison • Unexpected complex segmented relationship • Share a common 4.1 Mb ‘backbone’ or common, and generally colinear sequence (only 1 inversion) • Homologous sequences are interspersed with HUNDREDS of ISLANDS of INTROGRESSED DNA Dr M. D-S, 2007

  46. E.coli O157:H7 - K12 genome comparison • The specific DNA segments for each strain were named ‘O islands’ , ie O157:H7-specific DNA segments, or ‘K islands’ • Backbone of 4.1 Mb common sequence. Not identical (eg 75% of proteins differ by one aa). • O-islands total 1.34 Mb (about 26% of genes !) • Largest O-island is 106 gene region (not small!) Dr M. D-S, 2007

  47. E.coli O157:H7 - K12 genome comparison • Virulence genes do not seem to be concentrated in one particular ‘island’; appear to be several • Often (189 cases), the backbone-island junction is WITHIN an ORF. O-island AUG UGA Protein coding ORF What does this pattern suggest? Dr M. D-S, 2007

  48. E.coli O157:H7 - K12 genome comparison • Suggests that incoming DNA recombined with the genome (somehow?) rather than inserted. O-island AUG UGA Protein coding ORF Dr M. D-S, 2007

  49. Comparative Genome Map Dr M. D-S, 2007

  50. Distribution of O-islands of EDL933 specific sequence (red), ‘K-islands’ of K12 specific sequence (green) and common ‘backbone’ sequence (blue) Genome Map GC-content of genes, plotted around mean GC-skew for 3rd codons Scale, in base pairs Octamer Chi sequences Dr M. D-S, 2007

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